The successful monitoring of an estrous (also known as oestrous) cycle has many potential uses. For example, artificial insemination of animals, especially cattle and other livestock, as well as other mammals is often employed by dairy farmers and other animal caretakers for breeding. Successful impregnation requires that the artificial insemination procedure take place at the appropriate period within the animal's estrous cycle. Additionally, successful monitoring can allow breeding to be timed to coincide with the appropriate period of an animal's estrous cycle. Such timing may be especially important when, for example, a mare is to be transported to a stallion for breeding. Another example of a potential use for monitoring an estrous cycle is in connection with laboratory testing and studies. Accuracy of certain tests may depend on the administration of a drug or the performance of another procedure during a particular phase of the estrous cycle, or synchronization of testing on test animals so that the administration procedure is performed at the same phase of the subjects' estrous cycles.
The estrous cycle of a female mammal that reabsorbs the endometrium, in contrast to menstruation which releases the endometrium, as occurs in primates, involves recurring hormone-induced physiological changes and generally is characterized by four phases: proestrus, estrus, metestrus, and diestrus. During proestrus, prostaglandin PF2α causes regression of the corpus luteum developed in the previous cycle. As the corpus luteum is destroyed, there is a fall in progesterone levels. The fall in progesterone levels is accompanied by an increase in the production of follicle stimulating hormone (FSH), which stimulates follicular growth in the follicle that will result in ovulation. During this phase there is an increase in circulating estradiol levels.
The estrus phase refers to a periodic state of the estrous cycle in mammals that do not experience menstruation. The estrus phase has two stages. The first stage of the estrus phase is initiated as estradiol levels rise and cause the production of luteinizing hormone (LH). During this first stage of the estrus phase, which is also known as behavioral estrus or “heat,” the estradiol levels will begin to decrease as the LH level surges to a maximum concentration or LH peak. The length of the behavioral expression of estrus (or “heat”) varies from animal to animal. An example of a female mammal with spontaneous ovulation is the cow. Generally, subject to variations between individual animals, behavioral estrus in a cow lasts between 8-28 hours. The subsequent second stage of the estrus phase runs from the LH peak to ovulation. In cows, ovulation may occur approximately 12-18 hours after behavioral estrus or “heat” has ended. During this second stage of the estrus phase, successful inducement of pregnancy is most likely to occur.
Metestrus is a period of sexual inactivity following the estrus phase. Metestrus can last from 1-5 days as observed in the case of cows. In metestrus, early corpus luteum development begins anew in a process known as luteinization and progesterone levels begin to rise. Estradiol levels increase in cyclic phases during metestrus. Metestrus lasts until the beginning of the diestrus phase. During diestrus, estradiol varies in cyclic waves of about 4 days duration and levels of estradiol remain relatively low until proestrus, when the corpus luteum is destroyed through the action of prostaglandins such at PF2α which causes progesterone to fall and estradiol levels to increase to their maximal levels. The cycle thereby repeats itself.
Estrous cycle frequency and duration varies from species to species. Some species of mammals have spontaneous ovulation that comes in regular cycles. The estrous cycles of some species with spontaneous ovulation can also be seasonal. Some species have only one “heat” per season, while others may have multiple heats. Other types of mammal species have induced ovulation which is stimulated by the presence or contact with a male of the same species. Examples include rabbits, camels, and alpacas.
Failure to timely inseminate during the appropriate phase of the estrous cycle, preferably immediately before ovulation, creates a significant economic burden to the farmer. For seasonal breeders, for example, unsuccessful breeding can cause the breeder to wait weeks or months for another breeding opportunity. Further, the insemination and breeding procedures are themselves expensive, and repeating the procedures multiple times on the same animal for a single successful pregnancy can significantly increase costs. Furthermore, the delay inherent in waiting for the next estrus phase or seasonal estrous cycle to re-inseminate the animal compounds the economic burden on the breeder, especially if the animal produces milk, for which maximal production may be dependent on successful breeding and a continuous stream of pregnancies.
About half of all cow estrus phases fail to be observed because the farmer is either not present to actually observe the animal in estrus or because existing estrus detection tests are not sufficiently reliable. P. L. Senger, Estrus Detection Problem New Concepts Technologies and Possibilities, J. Dairy Science, 77:2745-2753 (1994). It has been estimated that failed insemination and breeding costs U.S. dairy farmers over $300 million annually. R. L. Wallace, Economic Efficiencies of Dairy Herd Reproductive Programs, DVM, MS Illinois Dairy Net Papers (Mar. 13, 2002). Hence, accurate and reliable detection of the estrus phase is highly important for high impregnation and breeding success rates and, ultimately, is highly important to farmers and other breeders for economic reasons.
Anthocyanin pigments can be used to measure fertility and estrogen-dependent physiological changes in females. U.S. Pat. Nos. 4,358,288, 5,922,613, and 5,981,291 describe the color response that an anthocyanin pigment produces when contacted with a body fluid such as saliva or vaginal fluid. It has now been observed by the present inventor that certain forms of anthocyanin pigments specified in the aforementioned patents, specifically 3,5-diglycosidyl anthocyanins, show near identical color responses to the unaided eye for both the fertile estrus phase and the mid-luteal phase (i.e., diestrus) of the estrous cycle when tested on a cellulose surface with no other agents. Because the likelihood of successful pregnancy when insemination or breeding in the mid-luteal phase is significantly lower than in the fertile estrus phase, it is desirable for an estrus phase detection test to be capable of distinguishing between these phases.
Commercial kits are available for estrus evaluation of female livestock and other mammals. These known commercial kits at best identify only the general phase of estrus; they do not distinguish between the first stage of the estrus phase (before the LH peak) and second stage of the estrus phase (after the LH peak). The second stage of the estrus phase is the optimal time period for insemination and breeding. Timing insemination or breeding to coincide with the second stage of the estrus phase is important for optimum pregnancy results. Insemination and breeding in the first stage of the estrus phase does not generate a pregnancy at nearly the rate of breeding as in the second stage of the estrus phase. Lack of clarity and consistency in known commercial kits results in inaccurate timing for insemination and breeding, causing lower pregnancy rates, increased costs, and decreased efficiency to the breeder.
Accordingly, it is highly desirable to have an estrus detection procedure that allows the inseminator/breeder to determine the estrus phase, and more desirably distinguish the optimal fertile stage of the estrus phase from other phases of the estrous cycle, including the mid-luteal and diestrus phases and desirably the first stage of the estrus phase, in order to efficiently and effectively determine whether the female is (or when the female will be) ready for insemination/breeding.